Difference between revisions of "Part:BBa K2332040"

Latest revision as of 14:36, 25 October 2017

Blue light inducible expression of Intimin'-GFP-SpyTag (Pblind Intimin'-GFP-SpyTag)

This construct displays SpyTag fused to GFP on the cell surface of E. coli cells under the control of Pblind, a blue light inducible promoter. Cells must also express EL222 (BBa_K2332004). Upon blue-light (465nm) exposure, EL222 dimerises and binds to its DNA-binding region within Pblind to recruit RNAP and induce transcription of Intimin'-GFP-SpyTag.

Intimin' is a truncated version of Intimin (EaeA intimin), an outer membrane surface protein, which has shown to display proteins of similar size to GFP fused to its N terminal. This construct allows to directly observe through fluorescence microscopy the functionality of Intimin for cell surface display under blue light induction.

This construct can be used in combination with SpyCatcher constructs. SpyTag (13 amino acids) and SpyCatcher (138 amino acids, 15 kDa) are protein binding partners that form spontaneous irreversible isopeptide bonds between an aspartate residue in SpyTag and a lysine residue in SpyCatcher. These binding partners originate from CnaB2 (immunoglobulin-llike collagen adhesin domain) of the FbaB protein, found in the invasive strains of S. pyogenes. Thus, this construct allows to directly observe through fluorescence microscopy SpyTag localization on the cell surface, and most importantly its co-localization with SpyCatcher variants. Particularly for our project, it would allow us to directly observe cell-cell binding post blue-light exposure when used in combination with cells expressing intimin'-SpyCatcher.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal SpeI site found at 2844
12
INCOMPATIBLE WITH RFC[12]
Illegal SpeI site found at 2844
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal SpeI site found at 2844
25
INCOMPATIBLE WITH RFC[25]
Illegal SpeI site found at 2844
Illegal NgoMIV site found at 880
Illegal NgoMIV site found at 1621
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 2715

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 <partinfo>BBa_K2332040 short</partinfo>
-This construct displays GFP fused with SpyTag on the cell surface of E. coli cells under the control of our blue light inducible promoter. Intimin' is a truncated version of Intimin (EaeA intimin), an outer membrane surface protein, which has shown to display proteins of similar size to GFP fused to its N terminal. This construct allows to directly observe through fluorescence microscopy the functionality of Intimin for cell surface display under blue light induction.
+This construct displays SpyTag fused to GFP on the cell surface of E. coli cells under the control of [https://parts.igem.org/Part:BBa_K2332002 Pblind], a blue light inducible promoter. Cells must also express EL222 ([https://parts.igem.org/Part:BBa_K2332004 BBa_K2332004]). Upon blue-light (465nm) exposure, EL222 dimerises and binds to its DNA-binding region within Pblind to recruit RNAP and induce transcription of Intimin'-GFP-SpyTag.
+Intimin' is a truncated version of Intimin (EaeA intimin), an outer membrane surface protein, which has shown to display proteins of similar size to GFP fused to its N terminal.
+This construct allows to directly observe through fluorescence microscopy the functionality of Intimin for cell surface display under blue light induction.
+This construct can be used in combination with SpyCatcher constructs. SpyTag (13 amino acids) and SpyCatcher (138 amino acids, 15 kDa) are protein binding partners that form spontaneous irreversible isopeptide bonds between an aspartate residue in SpyTag and a lysine residue in SpyCatcher. These binding partners originate from CnaB2 (immunoglobulin-llike collagen adhesin domain) of the FbaB protein, found in the invasive strains of S. pyogenes.
+Thus, this construct allows to directly observe through fluorescence microscopy SpyTag localization on the cell surface, and most importantly its co-localization with SpyCatcher variants. Particularly for our project, it would allow us to directly observe cell-cell binding post blue-light exposure when used in combination with cells expressing intimin'-SpyCatcher.
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